This invention relates to an improvement in rapid and highly sensitive assays for ligands contained, or suspected of being contained, in liquids of non-uniform character in which solid particles and/or other undissolved substances of a semisolid or colloidal nature that are likely to interfere with or obscure the result of the assay typically are also present. Such liquids include, e.g., blood, urine, lymph, lacteal fluid, and like biological liquids and liquids that have been deliberately constituted such as inoculated bacterial media, dilutions thereof, dilutions of biological samples, etc., and non-biological liquids that contain solids, semisolids or colloids as collected such as groundwater and other environmental samples, sea water, surface water, potable water, liquid from heating, ventilation and air conditioning systems, waste water and like liquids. The assay may be an immunoassay performed in a disposable solid state device comprising a suitably housed test strip which is in turn comprised of sorbent material and which defines a flow path along which the liquid test sample travels to a test site impregnated with a conjugate of (1) a binding partner for the ligand sought to be detected, and (2) a particulate material that produces visible color in the presence of the ligand. The improvements achieved by using this invention can also be beneficially realized in assays other than immunoassays, such as receptor or molecular probe assays.
A test device suitable for immunoassay may be so constituted that it performs the assay in either the sandwich or the competition mode. Such a test device is often constructed so as to yield a quantitative result for the ligand, but it may also be designed and constructed so as to furnish only a positive or negative answer concerning the presence of the ligand. Its flow path may be designed for unidirectional or bidirectional flow of components.
The assay in which the benefits of this invention are realizable must be extremely sensitive, so that it is able to detect very low concentrations of ligand in the test fluid accuratelyxe2x80x94i.e., without producing the false positive or false negative results often experienced when using both currently sold disposable solid state devices and prior art assay methods which required multiple dilutions of the biological fluid. The latter assays in the prior art often employed, e.g., xe2x80x9cdipstickxe2x80x9d type devices and the like and required relatively long incubation times for reaction of the ligand with its labeled binding partner. Obviously because the test fluids referred to above typically contain solid and semisolid particles and/or other undissolved components such as fats and other colloidal substances that may interfere with or obscure the result of an assay for a particular dissolved ligand, it is important to provide for removal of these potential interferants prior to the performance of the assay itself.
U.S. Pat. No. 5,714,389 issued Feb. 3, 1998 to Charlton et al. describes assays performable on bodily fluids containing solid particulate material using specially constructed test devices which are adapted to be dipped directly into the fluid. In these devices the sample flow path is constructed so as to include, near its ingress, a filtration means having the purpose to trap particulate components of the liquid test sample as that sample flows toward the test site. The patent alleges that the inclusion of this filtration means contributes to the sensitivity and accuracy of the test, and especially to the low incidence of false positives, including borderline false positives, observed from its use.
An important object of the present invention is to achieve all of the advantages of the test system disclosed in the Charlton et al. patent, including sensitivity and rapidity of the assay, low incidence of false positives and ability to yield reliable results even in the hands of untrained personnel, without experiencing the disadvantages of the specific test device therein described. In particular, an object of this invention is avoidance of the need for directly dipping the test device into the biological test fluid, coupled with avoidance of the need for interposing a filtration means in the flow path of the sample. By dispensing with such a filtration means, one avoids problems that are experienced in instances where the filter means becomes at least partially clogged, or even wholly clogged, by solid, semisolid and/or colloidal particles contained in the test fluid, making it necessary to discard the device without obtaining any useful assay result.
Another important object of this invention is to improve the reliability of delivery to an assay system that will benefit therefrom, whether of the immunoassay, molecular probe assay, receptor assay or some other assay type, of a relatively constant and reproducible sample volume to the test device.
The invention involves the use of a swab which contacts the test sample by immersion therein so that it is wetted, and preferably saturated, with the test sample liquid. The wetted swab is then removed from the sample and applied to a test device. In immunoassay applications, the sample applied to the test device is placed in contact with a test strip which has been designed and constructed to assay specifically for a particular ligand suspected of being present in the test liquid. The swab entraps solid and semisolid particulate matter and other undissolved matter such as fats and other colloidal material contained in the test sample and delivers only the liquid portion of the sample which is to be assayed into contact with the test strip. Typically, the sample then moves along a flow path, reacting with a binding partner that has been conjugated to a label. In cases where the test sample is, e.g., urine, many known test devices for home use require the user to contact the device with a urine stream. According to the present invention, the user will be instructed to contact the swab (which is supplied firmly attached to a handle) with the urine stream and then apply the swab to the sample device. Use of the swab thus eliminates all risk of device contamination with unrelated substances; it also minimizes the potential for contact of the urine by the user""s hands.
Preferably the swab is formed of a loose, fluffy fibrous material but other fibrous materials and certain open-pore materials, such as foamed resins and rubbers and sponges can also be used. Closed-cell materials that lack fibrous structure, however, are unsuitable for the swabs used in this invention. Swabs have been widely used heretofore to apply medicaments, to dry exposed surface areas of an inanimate object or a mammalian body, or to collect a small specimen of material to be viewed or further treated by moving the swab across a surfacexe2x80x94e.g., the xe2x80x9cthroat swabxe2x80x9d referred to in Example 1 of application Ser. No. 07/706,639 now U.S. Pat. No. 6,168,956 of Howard Chandler, assigned to SmithKline Diagnostics, Inc., but exclusively licensed in a wide area of applications to Binax, Inc., the intended assignee of the present application. Swabs have also been used heretofore to collect solid or semisolid samples in small quantitiesxe2x80x94e.g., phlegm, sputum, nasal discharge, etc.xe2x80x94for microscopic viewing or transfer to another receptacle for further processing.
According to this invention, the swab is to be immersed in the liquid sample, whereby liquid is sorbed by it and solid, semisolid and colloidal particles either cling to it or are left behind in the liquid. In contrast, swabs have been used in the past to collect samples by moving them across a semi-dry surface (such as the back of a patient""s throat) or touching them to a mass of semisolid matter. In this invention, when the swab, after immersion in the test liquid, is deposited in a partially covered swab well in accordance with this invention as hereinafter more fully described, the absorbed liquid sample is largely expelled, but the entrained solids, semisolids and colloids that cling to a fibrous swab surface or become trapped in the open-pore structure of a suitable non-fibrous swab are retained in or on the swab structure. While different types of swabs may vary somewhat in their capacity to absorb liquid upon immersion thereinxe2x80x94i.e., a cotton or Dacron swab and a sponge may have slightly different absorptive capacitiesxe2x80x94any given swab type of relatively uniform size has been found to absorb and to deliver to the test device an essentially uniform volume of liquid sample. By contrast, a pipette is not only easily clogged by solid, semisolid or colloidal substances, but in instances where the clogging is only partial, it may be hard to judge the liquid sample volume delivered to a test device, making it difficult in some cases to compare what should be duplicate results from a given test liquid. A similar problem, of course, plagues devices adapted to be dipped directly into the test sample and this problem exists whether or not the device is equipped with a filter means in the sample flow path as described in U.S. Pat. No. 5,714,389.
A test device preferably used for immunoassay in conjunction with the use of this invention is an immunochromatographic device of the type disclosed in application Ser. No.07/706,639 of Howard M. Chandler filed May 29, 1991, having two opposable, preferably hinged, halves wherein one half contains a sample well or sample pad and the other half carries the test strip comprising a flow path and test site and the liquid test sample is applied to the flow path by bringing the halves of the device into closed contact. This device can be adapted for use in a unidirectional or bidirectional flow assay format as therein disclosed; in either case, the improvement provided by the present invention can be readily utilized. The device may be specifically adapted to perform a sandwich or a competitive immuno-assay. It may also be specifically adapted to provide either a qualitative or a quantitative result.
Other immunoassay test devices wherein liquid sample is caused to move from the inlet end of a test strip along a flow path by means of capillary action, wicking, sorption or any other mechanism effective to create flow of sample along the strip have been described in the art and may also be readily adapted to receive a swab that has been wetted with test sample by immersion therein to apply test liquid therefrom to a test strip that has been suitably prepared to assay the test sample for the particular ligand to be determined. Examples of such immuno-assay devices include that disclosed by Charlton et al., which could readily be so modified and that disclosed, e.g., in May et al. U.S. Pat. No. 5,622,871 issued Apr. 22, 1997, which likewise could be readily adapted to receive a swab previously immersed in a test sample that performs the dual function of applying liquid test sample to the flow path and filtering out solid particles and other undissolved substances which might potentially interfere with the assay.
Among the ligands which can successfully be assayed for using this method are bacteria such as E. coli, various lower respiratory tract organisms such as those that cause pneumonia, e.g., Streptococcus or Legionella, various upper respiratory tract organisms such as Hemophilus and those that cause influenza-like syndromes, sexually transmitted bacteria such as those that cause gonorrhea, antigens such as human chorionic gonadotropin (hCG), spermatozoa antigens, human luteinizing hormone, progesterone and estrogen. Other suitable ligands detectable by this method will readily occur to those skilled in the art.
The immunoassay may be of the xe2x80x9csandwichxe2x80x9d or the xe2x80x9ccompetitivexe2x80x9d type, depending upon the particular ligand to be assayed for. In all cases, it is performed in a manner that is generally described in the art, e.g., in application Ser. No. 07/706,939 and in various patents such as those of Charlton et al. and May et al. cited hereinabove. The other types of assays, such as receptor and molecular probe assays, to which the invention is applicable, may likewise be configured in various formats, all of which can readily be adapted to receive sample delivered by a swab.